Because substantially greater peak capacity and resolution can be obtained by 2-dimensional separation systems, a variety of 2-dimensional and coupled separation mechanisms have been developed. The coupling of liquid chromatography and gas chromatography to mass spectrometry, MS-MS, LC with electrochemical detection, coupled column techniques and other such systems can produce a wealth of information on complex samples. In the article "Comprehensive 2-Dimensional HPLC" (Bushey, M. and Jorgenson, J., Anal. Chem. 1990, 62, 161-167) a method was presented which coupled ion exchange chromatography and size exclusion chromatography in a manner such that all of the effluent from the first column was reanalyzed on the second column without the use of stopped flow methods.
Many samples need to undergo more than one separation mechanism to reduce peak overlaps (Davis, J. and Giddings, J., Anal. Chem. 1985, 57, 2168-2177; Davis, J. and Giddings, J., Anal. Chem. 1985, 57, 2178-2182). For two techniques to be satisfactorily coupled, however, several criteria need to be addressed. Most importantly, the two techniques should be as orthogonal to each other as possible (i.e., the two techniques should base their respective separations on as different sample properties as possible). The orthogonality aspect creates an interesting problem in the design of 2-D separation systems; the more orthogonal two separation mechanisms are, the more dissimilar they will be in operation, and the more dissimilar the two systems are in operation, the more difficult it will probably be to couple the two systems.
Reversed phase chromatography (RP HPLC) and capillary zone electrophoresis (CZE) are presumably highly orthogonal separation methods. Based on this, they are good candidates for pairing in a 2-D system. Several groups have already recognized this and have used CZE to analyze collected RP HPLC fractions of enzymatic digests of proteins and to compare the tryptic digest fingerprints of RP HPLC and CZE (Puma, P. et al., poster M-P-126 presented at HPCE '89, Boston, Mass., Apr. 10-12, 1989; Nielsen, R. et al., J. Chromatogr. 1989, 480, 393-40; Grossman, P. et al., Anal. Chem 1989, 61, 1186-1194). The unsurprising result in these cases is that CZE is found to be able to resolve some peptides that co-elute on RP HPLC. Although no one has actually reexamined collected CZE fractions by RP HPLC, by careful examination of RP HPLC and CZE tryptic digest maps of human growth hormone, Nielsen and coworkers (J. Chromatogr. 1989, 480, 393-40) have shown that the opposite is true also; that RP HPLC is capable of resolving peptides that co-migrate in CZE. This is not a surprising result, especially considering those species with zero net charge that migrate at the velocity of the electroosmotic flow and are thus unresolved by CZE. Pairing electrophoretic separations with chromatography has also been done using isotachophoresis as a purity check for RP HPLC analysis of peptides (Janssen, P. et al., J. Chromatogr. 1989, 470, 171-183).
Only one other group that we are aware of has attempted to automate the coupling of HPLC with CZE. That attempt coupled a Sephadex G-50 size exclusion column as the first dimension separation to isotachophoresis as the second dimension separation. Second dimension analysis times, however, were very long at 18 minutes each, stopped flow methods were used on the first column during the second dimension separation, and 3-D "chromatoelectropherograms" were not presented. The system was used to analyze a sample containing bovine serum albumin, myoglobin and tyrosine (Yamamoto, H. et al., J. Chromatogr. 1989, 480, 277-283). There is, accordingly, a clear need for new 2-D analysis systems